Mersacidin belongs to a group of bactericidal peptides that are called lantibiotics. The name signifies that these peptides contain the amino acids lanthionine and/or 3-methyllanthionine. Mersacidin has activity against methicillin-resistant Staphylococcus aureus (MRSA) and is therefore of considerable interest in medicine.
Mersacidin is produced by a specific species of the genus Bacillus, which has been designated HIL Y-85,54728 (“HIL”). The cloning of the mersacidin gene is disclosed by Bierbaum et al, 1995.
Mersacidin is produced by processing of a small protein of 68 amino acids. The N-terminal 48 amino acids of the protein form a leader sequence, and the C-terminal 20 amino acids are a propeptide sequence which is processed by modifying enzymes to produce mersacidin. The sequence of the mersacidin gene, mrsA, is provided as SEQ ID NO:1 and its translation as SEQ ID NO:2.
The mrsA gene forms part of the mrs gene cluster of about 12.3 kb (Altena et al, 2000). The gene cluster includes regulatory genes which control the production of mersacidin by regulating the expression of the mrsA gene and/or its modifying enzymes. The mrsA gene is expressed in early stationary phase of the growth of the Bacillus HIL strain.
A problem with the use of Bacillus HIL as a host cell for the production of products of interest is the fact that under certain conditions the host cell sporulates. For larger scale production presence of spores potentially causes significant handling difficulties especially if the producer strain is a GMO, as is likely to be the case for a producer of a variant mersacidin. In case of a spillage, bacillus spores are difficult to kill with most disinfecting chemicals. Removal of spores in process streams would be difficult without expensive microfiltration. A sporulating GMO is therefore likely to require a higher cost and complexity of engineering for containment and processing.
At a research level, the presence of spores can make the development of alternative lantibiotics based upon engineering of the wild-type gene cluster difficult. For example, overlay assays for anti-bacterial activity can be spoiled by outgrowth of spores.
A further problem generally with the Bacillus HIL strain is that it—in common with many other Bacillus strains—produces other products with anti-bacterial activity. These products can interfere with the development of assays designed to investigate the properties of variant Bacillus HIL strains.
Sigma H is the product of the sigH (or spo0H) gene. It is essential for transcription of genes that function in the transition from exponential to stationary phase and in the induction of sporulation. Mutants deficient in SigH do not sporulate. SigmaH activates transcription of a number of other regulatory proteins e.g. spo0A, spo0F, kinA, spo0M, spoVG, spoVS and the spoIIA family as well as the phr family of secreted peptide pheromones. For further details see Britton et al. J Bacteriol. 184, 4881-90; 2002.
Directly or indirectly Sigma H influences transcription of about 10% of all genes of Bacillus (Britton et al., 2002). Early results showed that sigma H is involved in the biosynthesis of gramicidin S (Marahiel et al. Mol. Microbiol. 7, 631-636; 1993), and Britton et al. found that the following antibiotic production genes are downregulated in a sigH deficient mutant: cotN (tasA, antimicrobial spore component), pksCDEFGHIKLMNR (polyketide synthesis), pnbA (paranitrobenzyl esterase), srfAB (surfactin synthetase), ywhP (albD) and ywiA (albA, both involved in antilisterial bacteriocin production), thus knockout affects multiple antibiotic biosynthesis pathways.
Szekat et al. (2003) Appl. Env. Microbiol. 69, 3777-3783 describe the construction of an expression system for generation of variant mersacidins. Modified mrsA genes are generated by site-directed mutagenesis using a commercial phagemid system. The modified genes are then excised and ligated into a temperature sensitive plasmid which replicates in Gram-positive bacteria such as Bacillus sp. The plasmids are introduced into Staphylococcus carnosus by protoplast transformation and then introduced into the mersacidin-producing bacillus again by protoplast transformation. The bacilli are then grown at elevated temperature so that the plasmid cannot replicate autonomously and thus integrates into the chromosome by homologous recombination in the mrsA region. At this stage the bacillus now contains the entire expression plasmid inserted into the mersacidin biosynthetic pathway and hence has two copies of the mrsA gene, one of which is mutated and the other wild-type.
These constructs do not produce either mersacidin or the engineered variant presumably due to disruption of other elements of the biosynthetic pathway. The next stage is to grow these constructs for a large number of generations without selection for the plasmid in order to allow a second recombination event to occur to excise the plasmid and to leave a single copy of the mrsA gene. Depending on where the recombination events occur this can either reconstruct the wild-type mrsA gene or generate the engineered variant and clones need to be screened to identify one in which the desired event has occurred. The net result is a direct replacement of the wild-type mrsA gene by a mutant gene in the chromosome. This procedure is lengthy and relatively inefficient for the production of large numbers of variants of mersacidin.
Three variants of mersacidin were produced by Szekat et al. (ibid); F3L, S16I and E17A (where the numbers refer to the numbering of the mature mersacidin peptide sequence and the letters are the 1-letter amino acid code).
Of these three variants, both the S16I and E17A were essentially inactive (about 1,000-fold greater Minimum Inhibitory Concentration (MIC) measured against M. luteus) while the F3L peptide was weakly active (MIC of 12.5 mg/l against M. luteus, compared to wild-type of 0.195). The data of Szekat et al thus suggest that mersacidin is very sensitive to alterations and variation of the primary sequence is likely to be deleterious.
Another problem associated with the production of mersacidin variants is that the Bacillus HIL has only been transformable at low frequencies using protoplast transformation. In order to investigate large numbers of mersacidin derivatives, a more efficient transformation system is required.
Variants of naturally occurring antibiotics can be useful in medicine. Variants can be produced synthetically, semi-synthetically (e.g. by chemical modification of fermented products) or by genetic changes to the organisms which produce them. Potentially, mersacidin could be varied by all three routes, with the latter two being of particular interest. For example, modification of amino acids could be used to produce variants which have altered activity profiles, as well as properties such as bioavailability, biodistribution or the ability to overcome resistance mechanisms to mersacidin itself. Altered amino acids may also be useful to introduce reactive side-chains allowing modification of the peptide by chemical means.